ABSTRACT Traditionally, viral vaccines have involved trade-offs between safety and immunogenicity, which is especially challenging for vaccines needed to control explosive emerging diseases where rapid protection and durable immunity are crucial. To overcome these challenges, we developed a mosquito-specific alphavirus, Eilat virus (EILV), as a revolutionary new vaccine vector. Eilat replicates to exceptionally high titers in mosquito cells but is completely defective for replication in vertebrate cells due to fundamental restrictions in entry and RNA replication. We generated a chimeric EILV cDNA with envelope proteins derived from chikungunya virus (CHIKV), which is responsible for major reemerging outbreaks of debilitating, chronic arthralgia involving millions of persons and recently affecting nearly all countries in the Americas. A CHIKV vaccine was recently ranked the #2 priority for global vaccine needs. In preliminary studies, the chimeric EILV/CHIKV vaccine candidate provided rapid and durable protection following a single dose against challenge of mice and nonhuman primates. We will further develop this chimera and test the hypothesis that EILV-based chimeric viruses can serve as ?pseudoinactivated? alphavirus vaccines, using three Aims: 1. Optimize the replication and immunogenicity of the chimeric EILV/CHIKV vaccine, confirm its safety (including unambiguously demonstrating a lack of RNA replication in vertebrate cells), verify efficient production in serum-free media, and develop inexpensive purification methods. 2. Study the protective efficacy of the optimized EILV/CHIKV vaccine in mice and understand the immune mechanisms of host protection, including characterization of innate and adaptive immune responses in vaccinated and challenged mice, and determination of the immune correlates of host protection. 3. Optimize the dosage of the EILV/CHIKV vaccine and confirm its rapid and long-lived immunogenicity and protection in cynomolgus macaques. These outcomes will be compared to inactivated and live-attenuated CHIKV vaccines. The result will be a safe, efficacious, inexpensive single-dose CHIKV vaccine that provides rapid and long-lived protection, as well as a platform technology that can be applied to all other pathogenic alphaviruses such as Venezuelan and Eastern equine encephalitis viruses, and the recently emerging Mayaro virus. These vaccines will be ideally suited for controlling explosive outbreaks that typically affect resource-poor tropical countries. The mechanistic studies to understand the remarkable immunogenicity of this vaccine will also impact other vaccine design platforms by dissecting the fundamental components of a viral vaccine required to induce various arms of the protective immune response.